Involvement of EphB1 receptors signalling in models of inflammatory and neuropathic pain.

EphB receptors tyrosine kinases and ephrinB ligands were first identified as guidance molecules involved in the establishment of topographical mapping and connectivity in the nervous system during development. Later in development and into adulthood their primary role would switch from guidance to activity-dependent modulation of synaptic efficacy. In sensory systems, they play a role in both the onset of inflammatory and neuropathic pain, and in the establishment of central sensitisation, an NMDA-mediated form of synaptic plasticity thought to underlie most forms of chronic pain. We studied wild type and EphB1 knockout mice in a range of inflammatory and neuropathic pain models to determine 1), whether EphB1 expression is necessary for the onset and/or maintenance of persistent pain, regardless of origin; 2), whether in these models cellular and molecular changes, e.g. phosphorylation of the NR2B subunit of the NMDA receptor, increased c-fos expression or microglial activation, associated with the onset of pain, are affected by the lack of functional EphB1 receptors. Differences in phenotype were examined behaviourally, anatomically, biochemically and electrophysiologically. Our results establish firstly, that functional EphB1 receptors are not essential for the development of normal nociception, thermal or mechanical sensitivity. Secondly, they demonstrate a widespread involvement of EphB1 receptors in chronic pain. NR2B phosphorylation, c-fos expression and microglial activation are all reduced in EphB1 knockout mice. This last finding is intriguing, since microglial activation is supposedly triggered directly by primary afferents, therefore it was not expected to be affected. Interestingly, in some models of long-term pain (days), mechanical and thermal hyperalgesia develop both in wild type and EphB1 knockout mice, but recovery is faster in the latter, indicating that in particular models these receptors are required for the maintenance, rather than the onset of, thermal and mechanical hypersensitivity. This potentially makes them an attractive target for analgesic strategies.

Nociceptor-expressed ephrin-B2 regulates inflammatory and neuropathic pain.

BACKGROUND: EphB receptors and their ephrin-B ligands play an important role in nervous system development, as well as synapse formation and plasticity in the adult brain. Recent studies show that intrathecal treatment with EphB-receptor activator ephrinB2-Fc induced thermal hyperalgesia and mechanical allodynia in rat, indicating that ephrin-B2 in small dorsal root ganglia (DRG) neurons and EphB receptors in the spinal cord modulate pain processing. To examine the role of ephrin-B2 in peripheral pain pathways, we deleted ephrin-B2 in Nav1.8+ nociceptive sensory neurons with the Cre-loxP system. Sensory neuron numbers and terminals were examined using neuronal makers. Pain behavior in acute, inflammatory and neuropathic pain models was assessed in the ephrin-B2 conditional knockout (CKO) mice. We also investigated the c-Fos expression and NMDA receptor NR2B phosphorylation in ephrin-B2 CKO mice and littermate controls. RESULTS: The ephrin-B2 CKO mice were healthy with no sensory neuron loss. However, pain-related behavior was substantially altered. Although acute pain behavior and motor co-ordination were normal, inflammatory pain was attenuated in ephrin-B2 mutant mice. Complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia was halved. Formalin-induced pain behavior was attenuated in the second phase, and this correlated with diminished tyrosine phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor subunit NR2B in the dorsal horn. Thermal hyperalgesia and mechanical allodynia were significantly reduced in the Seltzer model of neuropathic pain. CONCLUSIONS: Presynaptic ephrin-B2 expression thus plays an important role in regulating inflammatory pain through the regulation of synaptic plasticity in the dorsal horn and is also involved in the pathogenesis of some types of neuropathic pain.

Tamoxifen-inducible NaV1.8-CreERT2 recombinase activity in nociceptive neurons of dorsal root ganglia.

To explore the function of genes expressed in adult mouse nociceptive neurons, we generated heterozygous knock-in mice expressing the tamoxifen-inducible Cre recombinase construct CreERT2 downstream of the Na(V)1.8 promoter. CreERT2 encodes a Cre recombinase (Cre) fused to a mutant estrogen ligand-binding domain (ERT2) that requires the presence of tamoxifen for activity. We have previously shown that heterozygous Na(V)1.8-Cre mice will delete loxP flanked genes specifically in nociceptive sensory neurons from embryonic day 14. We therefore used the same strategy of homologous recombination and mouse generation, substituting the Cre cassette with CreERT2. No functional Cre recombinase activity was found in CreERT2 mice crossed with reporter mice in the absence of tamoxifen. We found that, as with Na(V)1.8-Cre mice, functional Cre recombinase was present in nociceptive sensory neurons after tamoxifen induction in vivo. However, the percentage of dorsal root ganglion (DRG) neurons expressing functional Cre activity was much reduced (<10% of the number found in the Na(V)1.8-Cre mouse). We also examined Cre recombinase activity in sensory neurons in culture. After treatment with 1 muM tamoxifen for 48 h, 15% of DRG neurons showed Cre activity. Na(V)1.8-CreERT2 animals may thus be useful for single cell studies of the functional consequences of gene ablation in culture, but are unlikely to be useful for behavioral studies.

BDNF modulates sensory neuron synaptic activity by a facilitation of GABA transmission in the dorsal horn.

Topical application of brain-derived neurotrophic factor (BDNF) to the adult rat isolated dorsal horn with dorsal root attached preparation inhibited the electrically evoked release of substance P (SP) from sensory neurons. This effect of BDNF was dose dependent (EC(50) 250 pM) and reversed by the tyrosine kinase inhibitor, K-252a. BDNF-induced inhibition of SP release was blocked by the GABA(B) receptor antagonist CGP 55485 but not by naloxone. Acute application of BDNF significantly increased potassium-stimulated release of GABA in the dorsal horn isolated in vitro and this effect was blocked by K-252a. Intrathecal injection of BDNF into the rat lumbar spinal cord induced a short-lasting increase in hindpaw threshold to noxious thermal stimulation that was blocked by CGP 55485 and was associated with activation of ERK in dorsal horn. These data suggest that exogenous BDNF can indirectly modulate primary sensory neuron synaptic efficacy via facilitation of the release of GABA from dorsal horn interneurons.

A peripheral nervous system actin-binding protein regulates neurite outgrowth.

Difference cloning has identified a Villin-like mRNA transcript expressed selectively in peripheral sensory and sympathetic neurons. Pervin, the encoded 820-amino acid protein, has 60% identity with Villin and is the rat homologue of Advillin. Coimmunoprecipitation studies demonstrate that Pervin and actin interact in vivo. Transfection of COS-7 epithelial cell lines demonstrates colocalization of epitope-tagged Pervin with green fluorescent protein-actin and results in an increase in process formation. This effect is abolished when the putative actin-bundling headpiece of Pervin is deleted. Biolistic transfection of primary cultures of rat dorsal root ganglion sensory neurons also results in increased neurite outgrowth with FLAG-tagged Pervin. Deletion of the actin-bundling headpiece inhibits normal neurite growth. These data suggest that Pervin may play a significant role in regulating process outgrowth in peripheral neurons through a mechanism that involves the activity of an actin-bundling domain.